Figure 1.
PTP inhibition leads to enhanced activation of Stat3 in keratinocytes.
(A) Primary keratinocytes were cultured and irradiated with UVB at a dose of 800 J/m2. Cells were collected at the indicated time. For inhibition of PTP (lane 7, 180*), 100 µM of Na3VO4 was treated for 12 h before UVB irradiation. Cells were collected 3 h after UVB irradiation. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies specific for Stat3 and phosphorylated Stat3 (PY-Stat3). (B) 3PC keratinocytes were cultured with Na3VO4 for the indicated times (1, 3, 6, and 12 h) and then cells were exposed to UVB at a dose of 800 J/m2. One hour following UVB irradiation, cells were collected and processed for western blot analysis. Cells treated with vehicle only were harvested 1 h after UVB exposure (i.e. 13 h after vehicle treatment). (C) 3PC keratinocytes were cultured either in the presence or absence of Na3VO4 for the indicated time without UVB irradiation. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies specific for Stat3 and PY-Stat3. Con: control keratinocytes without UVB or vehicle.
Figure 2.
TC45, SHP1, and SHP2 rapidly dephosphorylates Stat3 after UVB irradiation in vitro mouse keratinocytes.
(A) Western blot analysis of TC45, SHP1, and SHP2 in response to UVB irradiation. Mouse primary keratinocytes were cultured and irradiated with UVB at a dose of 800 J/m2. Cells were collected at the indicated time and total cell lysates were isolated. (B) Expression analysis of PTPRT by RT-PCR. Mouse primary keratinocytes were cultured and irradiated with UVB at a dose of 800 J/m2. Cells were collected at the indicated time and total RNA was isolated. (C-F) 3PC keratinocytes were transfected with siRNA specific for TC-PTP, PTPRT, SHP1, or SHP2. Cells were collected at the indicated time after UVB irradiation (800 J/m2). Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies specific for Stat3, PY-Stat3. (C) Inhibition of TC-PTP expression by siRNA. (D) Inhibition of PTPRT expression by siRNA. (E) Inhibition of SHP1 expression by siRNA. (F) Inhibition of SHP2 expression by siRNA. a no UVB.
Figure 3.
TC45 primarily localizes to cytoplasm and translocates to the nucleus after UVB irradiation in keratinocytes.
(A and B) Primary keratinocytes were cultured and irradiated with UVB at a dose of 800 J/m2. Cells were collected at the indicated time. Cytoplasmic and nuclear lysates were separately isolated. (A) Western blot analysis of cytoplasmic phosphorylated Stat3, TC45, SHP1, and SHP2 after UVB exposure in keratinocytes. N: nuclear fraction, 0 h. LDH was used as a control for cytoplasmic fraction. Right: Relative expression levels of TC45 were quantified by densitometry. Results are the mean + standard deviation from three independent experiments. (B) Western blot analysis of nuclear phosphorylated Stat3, TC45, SHP1, and SHP2 after UVB exposure in keratinocytes. C: cytoplasmic fraction, 0 h. Lamin A/C was used as a control for nuclear fraction. Right: Relative expression levels of TC45 were quantified by densitometry. Results are the mean + standard deviation from three independent experiments. (C) Quantification of nuclear and cytoplasmic TC45 in keratinocytes without UVB irradiation. Values represent the percentage of the quantified signals (Land 1 and 7) shown in (B) relative to loading volume and total protein yield obtained from both nuclear and cytoplasmic fractions. Results are the mean + standard deviation from three independent experiments. (D) Immunofluorescence analysis of nuclear translocation of TC45 after UVB irradiation. Primary keratinocytes were cultured and irradiated with UVB at a dose of 800 J/m2. Keratinocytes were subjected to immunofluorescence analysis by using an anti-TC-PTP antibody. The nuclei were visualized by DNA staining with DAPI (blue). Right: Quantification of TC45 localization. Subcellular localization of TC45 was scored according to whether it was higher in the cytoplasm (C > N), evenly distributed between cytoplasm and nucleus (C = N), or higher in the nucleus (C < N) for 200 cells at each time point. Results are the mean + standard deviation from three independent experiments. (E) Western blot analysis of nuclear phosphorylated Stat3 and TC45 after UVB exposure in keratinocytes. 3PC keratinocytes were cultured in serum free keratinocyte growth medium or medium containing 4% FBS, and irradiated with UVB at a dose of 800 J/m2. Relative expression levels of TC45 and phosphorylated Stat3 after 20 min of UVB exposure were quantified by densitometry. Results are the mean + standard deviation from three independent experiments.
Figure 4.
Nuclear translocation of TC45 in response to UVB irradiation is independent of phosphatase activity in keratinocytes.
(A) 3PC and (B) HaCaT keratinocytes were transfected with plamid vectors expressing TC45-WT or TC45-D182A/Q262N. After 2 h of UVB irradiation (800 J/m2), keratinocytes were subjected to immunofluorescence analysis by using an anti-myc antibody to detect exogenous TC45 expression. The nuclei were visualized by DNA staining with DAPI (blue). Right: Quantification of TC45 localization. Results are the mean + standard deviation from three independent experiments.
Figure 5.
TC-PTP deficiency retards Stat3 dephosphorylation after UVB exposure.
(A) Western blot analysis of phosphorylated Stat3 in total cell lysates of TC-PTP-deficient primary keratinocytes. Wild-type and TC-PTP-deficient primary keratinocytes were cultured and irradiated with UVB at a dose of 800 J/m2. Cells were collected after 1 h of UVB irradiation. Western blot analysis of (B) cytoplasmic and (C) nuclear phosphorylated Stat3 in TC-PTP-deficient primary keratinocytes. Wild-type and TC-PTP-deficient primary keratinocytes were cultured and irradiated with UVB at a dose of 800 J/m2. Cells were collected after 1 h of UVB irradiation. Cytoplasmic and nuclear lysates were separately isolated. Relative levels of phosphorylated Stat3 were quantified by densitometry. Results are the mean ± standard deviation from three independent experiments.
Figure 6.
TC45, SHP1, and SHP2 cooperatively recover the level of phosphorylated Stat3 in response to UVB irradiation.
3PC keratinocytes were transfected with siRNA specific for TC-PTP, SHP1, and SHP2. Cells were collected after 1 h of UVB irradiation (800 J/m2). Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies specific for Stat3, PY-Stat3. Lane 1 and 2, No siRNA; Land 3 and 4, Inhibition of TC-PTP expression by siRNA; Lane 5 and 6, Inhibition of TC-PTP and SHP1 expressions by siRNAs; Lane 7 and 8, Inhibition of TC-PTP and SHP2 expressions by siRNAs; Lane 9 and 10, Inhibition of TC-PTP, SHP1, and SHP2 expressions by siRNA. Relative levels of phosphorylated Stat3 were quantified by densitometry. Results are the mean + standard deviation from three independent experiments.
Figure 7.
UVB-induced Stat3 dephosphorylation is an initial protective mechanism occurred against UVB exposure.
(A-B) Mouse primary keratinocytes were cultured and irradiated with UVB at a dose of 800 J/m2. Cells were collected at the indicated time and total cell lysates were isolated. (A) Western blot analysis of Stat3-mediated target proteins in response to UVB irradiation. (B) Western blot analysis of phosphorylated Stat1, Stat3 and Stat5 in response to UVB irradiation. (C-E) Mice (n = 3) were irradiated with UVB at a dose of 1,000 J/m2 and total epidermal extracts were isolated at the indicated time point after UVB irradiation. (C) Western blot analysis of phosphorylated Stat1, Stat3, and Stat5 in response to UVB irradiation. (D) Western blot analysis of Stat3-mediated target proteins in response to UVB irradiation. (E) Western blot analysis of Stat3-mediated target proteins in the epidermis of Stat3-deficient mice. Stat3-deficient mice (n = 3) were irradiated with UVB at a dose of 1,000 J/m2 and total epidermal extracts were isolated at the indicated time point after UVB irradiation. C: wild-type mice, untreated.